Food composition for improving intestinal function comprising dead cells of lactobacillus paracasei HY2782

Abstract

The present invention relates to a food composition comprising dead cells of Lactobacillus paracasei HY2782. The dead cells of Lactobacillus paracasei HY2782 of the present invention strengthens tight junctions, and thus can improve intestinal function.

Description

Food composition for improving intestinal function containing killed Lactobacillus paracasei HY2782 cells

[0001] The present invention relates to a food composition for improving intestinal function comprising killed Lactobacillus paracasei HY2782 cells.

[0002] Tight junctions are a type of cell junction that restricts the movement of substances between connected cells, forming bands around the cells. These tight junctions act as a barrier, allowing nutrients in while preventing toxic substances from leaking out of the small intestine. A strong barrier with low intestinal permeability strengthens the balance of microbes that determine gut health. However, if intestinal immunity is weakened due to stress or other factors, barrier permeability increases, reducing nutrient absorption and allowing toxins like viruses and bacteria to enter the intestines, potentially triggering various inflammations.

[0003] Lactic acid bacteria, when consumed, are beneficial to human health. Their diverse functions have been reported, including preventing constipation and diarrhea and boosting immunity. However, live lactic acid bacteria are sensitive to external factors such as heat and moisture, making long-term storage difficult. Furthermore, they are easily destroyed during digestion, requiring large amounts of lactic acid bacteria to be consumed for their full effects. Furthermore, these bacteria must reach the intestines to be effective. Therefore, research has focused on killed lactic acid bacteria. Compared to live bacteria, killed lactic acid bacteria are less susceptible to external influences, making product storage easier. They also have a longer shelf life. Furthermore, they can easily reach the intestines, making them more effective. However, the difficulty of establishing indicators for killed lactic acid bacteria is a drawback. Existing indicators for live bacteria have been measured using methods such as injection plate methods. However, this method has been difficult to apply to killed lactic acid bacteria, leading to unclear quality control standards. A previous study reported a patent for a live lactic acid bacteria strain that enhanced immune function using lipoteichoic acid, a cell wall component, as an indicator. However, most other killed lactic acid strains have struggled to establish indicators.

[0004] Therefore, there is a need for ongoing research on lactic acid bacteria that can enhance intestinal function while enabling the setting of indicators that can clearly measure the number of bacteria.

[0005] One aspect of the present invention provides a food composition for improving intestinal function comprising Lactobacillus paracasei HP7 dead cells.

[0006] One aspect of the present invention provides a food composition for improving intestinal function comprising killed Lactobacillus paracasei HY2782 cells.

[0007] Another aspect of the present invention is to provide a health functional food composition for improving intestinal function comprising killed Lactobacillus paracasei HY2782 cells.

[0008] Another aspect of the present invention provides a method for producing Lactobacillus paracasei HY2782 dead cells, comprising the steps of: a) culturing Lactobacillus paracasei HY2782 in a medium containing a sprouted oat extract; b) centrifuging the culture solution to recover Lactobacillus paracasei HY2782 cells and mixing them with PBS; c) heat-treating the Lactobacillus paracasei HY2782 cells mixed with PBS.

[0009] One aspect is to provide a food composition for improving intestinal function containing killed Lactobacillus paracasei HY2782 cells.

[0010] According to one specific example, the Lactobacillus paracasei HY2782 may be cultured in a medium containing a sprouted oat extract.

[0011] According to one specific example, the Lactobacillus paracasei HY2782 dead cells may include avenanthramide as an indicator substance.

[0012] According to one specific example, the avenanthramide may include one or more of avenanthramide A, avenanthramide B, and avenanthramide C.

[0013] According to one specific example, the improvement in intestinal function may be by strengthening tight junctions within the intestine.

[0014] Another aspect is to provide a health functional food composition for improving intestinal function containing Lactobacillus paracasei HY2782 dead cells.

[0015] Another aspect provides a method for producing Lactobacillus paracasei HY2782 dead cells, characterized by comprising the steps of: a) culturing Lactobacillus paracasei HY2782 in a medium containing sprouted oat extract; b) centrifuging the culture solution to recover Lactobacillus paracasei HY2782 cells and mixing them with PBS; c) heat-treating the Lactobacillus paracasei HY2782 cells mixed with PBS.

[0016] When a food composition containing Lactobacillus paracasei HY2782 dead cells according to the present invention is consumed, tight junctions can be strengthened, thereby improving intestinal function.

[0017] Figure 1 is a drawing showing the process for producing Lactobacillus paracasei cells cultured in a sprouted oat extract medium.

[0018] Figure 2 is a drawing showing the number of bacteria confirmed during the process of culturing and killing Lactobacillus paracasei standard strain and Lactobacillus paracasei HY2782 (hereinafter referred to as 'HY2782') strain in a sprouted oat extract medium (cultivation, concentration, and killing).

[0019] Figure 3 is a drawing confirming the avenanthramide content of the standard strain of Lactobacillus paracasei and the HY2782 dead cells.

[0020] Figure 4 is a diagram comparing the expression level of ZO-1, a barrier marker, when Lactobacillus paracasei strain and HY2782 cells were treated with LPS-treated Caco-2 small intestinal cells.

[0021] Figure 5 is a diagram comparing the expression level of Occludin, a barrier marker, when Lactobacillus paracasei standard strain cells and HY2782 cells were treated with LPS-treated Caco-2 small intestinal cells.

[0022] Figure 6 is a drawing confirming the change in barrier permeability when Lactobacillus paracasei standard strain and HY2782 killed cells were treated to Caco-2 small intestinal cells treated with LPS.

[0023] Figure 7 is a drawing comparing the intestinal cell morphology observed when Lactobacillus paracasei standard strain dead cells and HY2782 dead cells were treated with LPS-treated Caco-2 small intestinal cells.

[0024] One aspect of the present invention provides a food composition for improving intestinal function comprising killed Lactobacillus paracasei HY2782 cells.

[0025] In one specific example, the Lactobacillus paracasei HY2782 may be cultured in a medium containing a sprouted oat extract.

[0026] The above-mentioned dead bacteria are already dead and are not affected by acidity such as stomach acid, so most of them can reach the intestines.

[0027] In one specific example, the Lactobacillus paracasei HY2782 dead cells may include avenanthramide as an indicator substance.

[0028] In one specific example, the avenanthramide may include one or more of avenanthramide A, avenanthramide B, and avenanthramide C. The avenanthramide is a class of phenolic compounds known as polyphenols, which are commonly found in oats, particularly in the bran and hull portions of the grain, but have not been found in the dead cells of the strain.

[0029] According to one embodiment, the standard strain of Lactobacillus paracasei cultured in a medium containing the sprouted oat extract did not contain avenanthramide, unlike Lactobacillus paracasei HY2782 of the present invention.

[0030] In one specific example, the improvement in intestinal function may be strengthening the tight junctions within the intestines. The improvement in intestinal function may mean improving leaky gut syndrome. Strengthening the tight junctions within the intestines may improve leaky gut syndrome, which exhibits symptoms such as weakened tight junctions, allowing intestinal byproducts, toxins, antigens, etc. to enter the bloodstream and stimulate the body's immune system, thereby causing diseases related to weakened immunity, various infectious diseases, and autoimmune diseases, or worsening existing diseases.

[0031] The above improvement means any act of strengthening intestinal function or reducing the degree of symptoms of weakened intestinal function by administering the above composition to a subject.

[0032] According to one specific example, the food may be in the form of a beverage, pill, tablet, capsule, fermented milk, jelly, candy, or ice cream.

[0033] When the composition of the present invention is manufactured into a food composition, in addition to Lactobacillus paracasei HY2782 as an active ingredient, it may include ingredients commonly added during food manufacturing, such as proteins, carbohydrates, fats, nutrients, seasonings, and flavorings. Examples of carbohydrates include monosaccharides such as glucose, fructose, etc.; disaccharides such as maltose, sucrose, oligosaccharides, etc.; and polysaccharides such as dextrin, cyclodextrin, etc., and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents, natural flavoring agents [thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.)] and synthetic flavoring agents (saccharin, aspartame, etc.) can be used.

[0034] For example, when the food composition of the present invention is manufactured as a drink, in addition to the Lactobacillus paracasei HY2782 of the present invention, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, Eucommia extract, jujube extract, and / or licorice extract may be additionally included.

[0035] In addition, the food composition of the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc.

[0036] These components may be used independently or in combination, and the proportion of these additives may be selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the food composition of the present invention, but is not limited thereto.

[0037]

[0038] Another aspect of the present invention is to provide a health functional food composition for improving intestinal function comprising killed Lactobacillus paracasei HY2782 cells.

[0039] The contents related to the above Lactobacillus paracasei HY2782, dead cells, Lactobacillus paracasei HY2782 dead cells, avenanthramide, and improvement of intestinal function are the same as those described above.

[0040] There are no special restrictions on the types of the above health functional foods. Examples of foods to which the killed Lactobacillus paracasei HY2782 cells can be added include dairy products including meat, sausage, bread, chocolate, pizza, ramen, other noodles, gum, ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes. When formulated as a beverage, the liquid ingredients added other than the Lactobacillus paracasei HY2782 strain or both are not limited thereto, and may contain various flavoring agents or natural carbohydrates as additional ingredients, as in conventional beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, etc.), and polysaccharides (e.g., conventional sugars such as dextrin, cyclodextrin, etc.), and sugar alcohols such as xylitol, sorbitol, and erythritol. In addition, natural sweeteners [thaumarin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.)] and synthetic sweeteners (e.g., saccharin, aspartame, etc.) can be used as sweeteners.

[0041] In another embodiment, the health functional food of the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and enhancers (cheese, chocolate, etc.), pectic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the food composition of the present invention may contain fruit pulp for producing fruit and vegetable beverages. These ingredients may be used alone or in combination, and the proportion of these additives is generally selected in the range of 0.001 to 50 parts by weight per total weight of the composition.

[0042]

[0043] Another aspect of the present invention provides a method for producing Lactobacillus paracasei HY2782 dead cells, comprising the steps of: a) culturing Lactobacillus paracasei HY2782 in a medium containing a sprouted oat extract; b) centrifuging the culture solution to recover Lactobacillus paracasei HY2782 cells and mixing them with PBS; c) heat-treating the Lactobacillus paracasei HY2782 cells mixed with PBS.

[0044] The above culture may be cultured at 25°C to 40°C, preferably at 30°C to 37°C, and the culture time may be 12 to 36 hours, preferably 18 to 30 hours.

[0045] The above heat treatment may be at 110°C to 130°C for 10 to 30 minutes, and preferably at 120°C to 125°C for 12 to 18 minutes.

[0046] In the method for producing the above Lactobacillus paracasei HY2782 dead cells, a step of drying the heat-treated cells after the step of heat-treating the Lactobacillus paracasei HY2782 cells mixed with PBS may be further included.

[0047] The Lactobacillus paracasei HY2782 dead cells manufactured by the above manufacturing method may be cells containing avenanthramide.

[0048] Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are provided for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0049]

[0050] Example 1: Preparation of Lactobacillus paracasei cells cultured in sprouted oat extract medium

[0051] The method for producing killed Lactobacillus paracasei cells cultured in a sprouted oat extract medium is as shown in Figure 1. Specifically, 1% sprouted oat extract was added to MRS liquid medium, followed by sterilization. Lactobacillus paracasei was inoculated into the sterilized medium, and cultured at 30°C to 37°C for 24 hours. The cultured strain was centrifuged, and the recovered cells were diluted by mixing with an appropriate amount of PBS. The mixed strain was heat-treated at 121°C for 15 minutes to kill Lactobacillus paracasei, and then dried.

[0052] Lactobacillus paracasei strains used were Lactobacillus paracasei HY2782 strain and the standard strain Lactobacillus paracasei ATCC 25302.

[0053] In order to confirm whether the sterilization process of the strain was carried out well, the change in the number of viable cells according to each process step was confirmed as shown in Figure 2. The number of viable cells was measured by diluting the culture solution or concentrate of each step by 1 / 10 and adding 10 -7 10 inland -9 The number of bacteria was measured using MRS solid medium.

[0054]

[0055] Example 2: Analysis of avenanthramide content

[0056] The avenanthramide content of the manufactured Lactobacillus paracasei dead cells was quantified using HPLC. As a result, as shown in Fig. 3, no avenanthramide was detected in the dead cells of the standard strain of Lactobacillus paracasei, but avenanthramides A, B, and C were detected in the Lactobacillus paracasei HY2782 strain.

[0057]

[0058] Example 3: Confirmation of improvement in barrier markers of Lactobacillus paracasei HY2782 dead cells.

[0059] Caco-2 cells were seeded in 6-well plates at 10 6 The cells were divided to obtain the required number of cells and cultured for about 14 days to induce differentiation. Groups excluding the Normal group were treated with 10 μg / ml of LPS and cultured for 24 hours. Afterwards, except for the Control group, which is a negative control group, the dead cells of Lactobacillus paracasei HY2782 and the dead cells of the standard strain of Lactobacillus paracasei were added to 10 6 The cells were treated at a concentration of CFU / ml and cultured for 24 hours. The expression levels of tight junction genes (ZO-1, Occludin) in the cultured cells in each well were measured using rt-PCR.

[0060] As a result, as shown in Fig. 4, it was confirmed that the barrier marker ZO-1 was enhanced in both the Lactobacillus paracasei HY2782 cells and the Lactobacillus paracasei standard strain cells. However, as shown in Fig. 5, it was confirmed that the Occludin expression level was significantly increased only in the Lactobacillus paracasei HY2782 cells. Therefore, it was confirmed that the Lactobacillus paracasei HY2782 cells increased both the barrier enhancement marker genes Occludin and ZO-1.

[0061]

[0062] Example 4: Confirmation of barrier permeability

[0063] Caco-2 cells were cultured in 10 6Cultured Caco-2 cells (cells / ml) were placed on the intestinal chip as much as possible and cultured for approximately 5 days for differentiation. Except for the Normal group, the remaining wells were treated with 10 μg / ml of LPS for 24 hours to induce inflammation and barrier leakage. Each well was treated with 10 μg / ml of dead Lactobacillus paracasei HY2782 cells and the standard strain of Lactobacillus paracasei and cultured for 24 hours. The supernatants were removed from the apical and basal compartments, and 4 kDa FITC-dextran was diluted in fresh cell culture medium and applied to the apical compartment, while fresh medium without phenol red was added to the basal compartment. Every 30 minutes, 100 μl of medium from the apical and basal compartments was collected and replaced with fresh medium. The collected medium was measured for fluorescence intensity at an excitation wavelength of 485 nm and an emission wavelength of 525 nm using a microplate reader. Permeability was calculated by substituting the Papp (cm / s) value into the following permeability calculation formula.

[0064] [Mathematical Formula 1]

[0065]

[0066] As a result, as shown in Fig. 6, in the group treated with Lactobacillus paracasei HY2782 dead cells, the apparent intestinal permeability was lower, similar to the Normal group, confirming that the barrier was strengthened.

[0067]

[0068] Example 5: Observation of intestinal cell morphology

[0069] Cells sufficiently cultured in 6 wells were treated with 10 ug / ml of LPS for 24 hours in the remaining wells, excluding the Normal group, to induce inflammation and barrier leakage. Each well was inoculated with 10 Lactobacillus paracasei HY2782 dead cells and Lactobacillus paracasei standard strain dead cells. 6Cells were treated at a concentration of 100 cells / ml and cultured for 24 hours. The cultured cells were fixed, tight junctions were fluorescently stained, and cell images were observed using a confocal laser microscope.

[0070] As a result, as shown in Fig. 7, in the Control group treated only with LPS and the Lactobacillus paracasei standard strain (typestrain) treated group with LPS, the tight junctions were stained more faintly compared to the Normal group, whereas in the Lactobacillus paracasei HY2782 killed cell treated group, the tight junctions of cells were confirmed to be distinct, as in the Normal group. Therefore, it was confirmed that the Lactobacillus paracasei HY2782 killed cell can improve intestinal leakage by strengthening the barrier.

[0071]

[0072] The present invention has been described above, focusing on preferred embodiments thereof. Those skilled in the art will appreciate that the present invention can be implemented in modified forms without departing from its essential characteristics. Therefore, the disclosed embodiments should be considered illustrative rather than limiting. The scope of the present invention is set forth in the claims, not the foregoing description, and all differences within the scope equivalent thereto should be construed as being encompassed by the present invention.

[0073] [Accession number]

[0074] Name of depositor: Korea Center for Biological Resources (KCTC)

[0075] Accession number: KCTC13438BP

[0076] Date of acceptance: December 19, 2017

[0077]

Claims

1. A food composition for improving intestinal function containing dead cells of Lactobacillus paracasei HY2782 (Accession No.: KCTC 13438BP).

2. In paragraph 1, The above Lactobacillus paracasei HY2782 was cultured in a medium containing sprouted oat extract. Food composition for improving intestinal function.

3. In paragraph 1, The above Lactobacillus paracasei HY2782 dead cells contain avenanthramide as an indicator substance. Food composition for improving intestinal function.

4. In paragraph 3, The above avenanthramide comprises at least one of avenanthramide A, avenanthramide B, and avenanthramide C. Food composition for improving intestinal function.

5. In paragraph 1, The above improvement in intestinal function is to strengthen the tight junctions within the intestine. Food composition for improving intestinal function.

6. A health functional food composition for improving intestinal function containing Lactobacillus paracasei HY2782 dead cells. 7.a) A step of culturing Lactobacillus paracasei HY2782 in a medium containing sprouted oat extract; b) A step of centrifuging the culture solution to recover Lactobacillus paracasei HY2782 cells and mixing them with PBS; c) A step of heat-treating the Lactobacillus paracasei HY2782 cells mixed with the above PBS; A method for producing a dead Lactobacillus paracasei HY2782 cell, characterized in that it comprises:

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